MXPA97008753A - Procedure for preparation of breast ways of the amino acid alkylaminofenilo proparano - Google Patents

Abstract

Process for the preparation of an enantiomer form of 2-amino 3- (4-alkylamino phenyl) propanoic acid of general formula (Y), in which Alk represents an alkyl radical containing 1 to 2 carbon atoms, in the form (s) or ( r) as well as its salts, starting respectively from (L) phenylamine or (D) -phenylalanine, by nitration, then transformation of the nitro radical of the 4-notro phenylalanine obtained, of the general formula (II), in which R is a hydrogen atom or a protective radical, in an alkylamino radical, after the protection given the case of the amino function of the phenylalani

Description

PROCEDURE FOR THE PREPARATION OF BOTTOM WAYS OF THE PROPANIC ALKYLAMINOPHENYL ACID.

The present invention relates to a process for preparing an enantiomer form of 2-amino (alkylamino-4-phenyl) -3-propanoic acid of general formula. wherein Alk represents an alkyl radical containing from 1 to 2 carbon atoms and their salts.

The preparation of (2S) 2-amino 3- (4-methylamino phenyl) propanoic acid has been described in patent application WO 94/08014 by the action of an N-methyl transferase on p-amino- (L) -phenylalanine. However, it will not be industrially exploited because of very low yields.

The preparation of racemic 2-amino 3- (4-ylamino phenyl) propanoic acid has been described by B. L. MOLDAVER AND Z. V. PUSHKA-RLVA, Chem. Abstr. , 55, 22226 g, - in addition the specificity of this reaction does not guarantee the production REF: 25690 of the N-monomethyl component (sintering the diethyl p-dimethylaminobenzyl-N-acetylaminoamalonate, then crystallization is observed, the presence of a mixture of p-aminophenyl-alanine and its mono- and dimethyl derivatives on the mother liquor of crystallization.

It has been found that the enantiomeric forms of this acid can be prepared directly from (L) phenylalanine or from (D) -phenylalanine, depending on what it is desired to obtain, respectively the form (S) or ® of the acid, with high yields and good product quality.

According to the invention, the (L) or (D) phenylalanine, whose amine function is optionally protected, is nitrated, then the nitro radical of the 4-nitro phenylalanine obtained from the general formula. wherein R is a hydrogen atom or a protective radical, is transformed into an alkylamino radical, after the protection (if R is hydrogen) of the amino function of phenylalanine.

The nitration is carried out by the nitric acid in sulfuric medium, at a temperature between -10 and -20 ° C.

The protection of the amino radical is carried out according to known methods, by a protective radical R whose placement and elimination does not alter the rest of the molecule. Particularly, it is operated according to the methods described by T. W. GREENE, Protective Groups in Organic Synthesis, A. Whiley-Interscience Publication (1981) or by Me OMIE, Protective Groups in Organic Chemistry, Plenum Press (1973). preferably the amino radical is protected to the amide state by a radical R = acyl, more particularly by an acetyl radical; or the amino radical is protected to the carbamate state by an R-alkyloxycarbonyl radical.

The transformation of the nitro radical into an alkylamino radical is effected by alkylating reduction when the Alk radical is a methyl or ethyl radical, or by reduction, formylation of the obtained amine and subsequent reduction of the formylamino radical when the Alk radical represents methyl.

The alkylating reduction is carried out by hydrogenation, under hydrogen pressure in the presence of Raney nickel, operating in the methanol at a temperature comprised between 20 and 40 ° C and under a pressure comprised between 100 and 200 kPa.

The alkylation is carried out under hydrogen pressure by the addition of benzaldehyde after formaldehyde in the case that Alk is a methyl or ethanol radical in the case that Alk is an ethyl radical.

The removal of the protective radical of the amino and the elimination of the benzyl radical are carried out successively. When the amino protecting radical is an acetyl radical, the elimination is effected by treatment in aqueous acidic medium, particularly hydrochloric acid. When Alk is a methyl or ethyl radical, the removal of the benzyl radical is effected by hydrogenation under hydrogen in the presence of palladium on carbon in aqueous acid medium, particularly aqueous hydrochloric acid at a temperature between 50 and 60 ° C and under a pressure between 100 and 200 kPa. In this case, the phenylalanine derivative of the general formula (Y) is obtained in the form of a salt. Eventually it can be released from its salt according to the usual methods that do not alter the rest of the molecule.

When it is desired to obtain a phenylalanine derivative of general formula (Y) by which Alk is methyl by reduction then formylation, the reduction of the nitro radical is effected on the nitro-4-phenylalanine of general formula (II) whose amino function is protected by a radical R and whose acid function has been previously protected, operating in reducing medium as for example by treatment with zinc and ammonium chloride in a methanol-water mixture. The formylation of the amine obtained is carried out by the formic acid, according to the usual methods of reaction of an acid on an amine, which do not alter the rest of the molecule. Particularly, tet ahydrofurane is operated at a temperature between 0 and 25 ° C. The reduction of the formylamino radical thus formed is favorably effected by the boranes. For example, borane methyl sulfide is used in tetrahydrofuran at a temperature below 25 ° C.

The protection of the amine is carried out as described above. The protection of the carboxylic acid function is carried out according to known methods which do not alter the rest of the molecule and in particular by esterification. The removal of the protective radical from the amine and the protective radical of the acid are carried out simultaneously by treatment in aqueous acid medium, particularly hydrochloric acid. • The product of general formula (I) can be eventually purified by physical methods, such as crystallization or chromatography.

The product prepared according to the invention can be either directly obtained in the form of a salt or transformed into an addition salt with the nitrogenous bases according to the methods known per se. These salts can be obtained, for example, by the action of a metal base (for example alkaline or alkaline earth), ammonia or an amino on a product according to the invention in a suitable solvent such as an alcohol, an ether or water or exchange reaction with a salt of an organic acid. The salt formed is precipitated after the eventual concentration of its solution is separated by filtration, decantation or lyophilization.

As an example of salts, salts with alkali metals (sodium, potassium, lithium) or with alkaline-earth metals (magnesium, calcium), ammonium salt, salts of nitrogenous bases (ethanolamino, diethanolamino, trimethylamino) can be cited. , triethylamino, ethylamino, propylamino, diisopropylamine, NN-dimethylethanolamine, benzylamine, deciclohexylamine, N-benzyl, phenethylamino, NN "-dibenzylethylenediamine, diphenylenediamine, benzydrylamine, quinine, choline, arginine, lysine, leucine, dibenzylamine), so the salts of addition with mineral acids (hydrochlorides, bromides, sulfates, nitrates, phosphates) or organic acids (suscinates, furmarates, maleates, methanesulfonates, toluenesulfonates, isethionates, tartrates, acetates, propionates, citrates, or with the substitution derivatives of these components.

The enantiomeric form of the 2-amino 3- (4-alkylamino phenyl) propanoic acid thus prepared by the process according to the invention can be useful in chemical synthesis. Parculularly it can be used in the preparation of > biologically active products and more particularly in synergistin derivatives such as pristinamycin IB or the corresponding ethylamino derivative, operating from a strain of a streptogramin producing microorganism, possessing at least one genetic modification that affects the biosynthesis of a streptogramin precursor of the B Group; said mutant strain is cultivated in a suitable culture medium, supplemented with the product prepared according to the invention, as an original precursor other than that of biosynthesis is altered.

The strains put in place are from the strains that produce natural streptogramins (pristinamycin, virginamycin ...), mutated. Particularly the mutant strains possess one or more genetic modifications at the level of at least one of their genes involved in the biosynthesis of group B streptogramin precursors. This or these genetic modifications alter the expression of the mentioned gene, ie they return that gene, and if necessary, to another of the genes involved in the biosynthesis of precursors, partially or totally, incapable of being encoded by the natural enzyme involved in the biosynthesis of at least one precursor.

The following examples given by way of non-limiting illustrate the invention: Example 1. To a suspension of 200 g of (L) -phenylalanine in 16609 cm 3 of water at 20 ° C is added, with stirring, 340 cm 3 of triethylamine. The mixture is kept under stirring at a temperature close to 20 ° C for 30 minutes until complete solubilization then cooled to 5 ° C. To this solution, 126 cm 3 of acetic anhydride are added dropwise, keeping an internal temperature close to 5 ° C. At the end of the addition, the mixture is kept at 5 ° C for one hour then cooled to 0 ° C. 200 cm3 of a hydrochloric acid solution at 36 ° C are added dropwise to this solution, maintaining the temperature close to 0 ° C. At the end of the addition the suspension is stirred one hour at 0 ° C, then filtered. The white precipitate obtained is rinsed with 400 cm 3 of water, squeezed and then dried under reduced pressure (20 kPa) around 50 ° C. 227 g of 2-acetamido-3-phenyl propanoic acid (2S) is thus obtained in the form of a white solid which melts at 160 ° C.

Preparation of the acid 2-acetamido-3 (4-nitro phenyl) propanoic acid (2S). To a stirred solution of 32.0 g of 2-acetamido-3-phenylpropanoic acid (2S) in 46.1 cm3 of 90% sulfuric acid and cooled to -15 ° C, 6.4 cm3 of water are added dropwise. 100% nitric acid maintaining a temperature of the mixture close to -15 ° C. At the end of the addition, the solution is kept at -15 ° C for one additional hour then brought to 40 ° C, at which temperature 70.4 cm3 of water is added. At the end of the water addition, the solution is allowed to return to a temperature close to 20 ° C. The precipitate obtained is filtered, rinsed 2 times in 32 cm3 of water, then squeezed out under reduced pressure (20 kPa) around 50 ° C. thus, 18.2 g of 2-acetamido 3- (4-nitro phenyl) propanoic acid (2S) are obtained in the form of a white solid decomposing at 191 ° C.

Preparation of 2 - acetamido 3 - ((N-methyl-benzylamine) -4 phenyl) propanoic acid dihydrochloride (2S): To 5.50 g of Raney nickel, a solution of 5.00 g of 2-acetamido-3- (4-nitro phenyl) propanoic acid (2S) in 100 cm 3 of methanol is added at 20 ° C under stirring. The solution thus obtained is purged three times to the nitrogen then three times to the hydrogen before being placed under a hydrogen pressure of 100 kPa under vigorous stirring at a temperature close to 30 ° C for 2.5 hours. To the solution taken at 25 ° C, 2.90 cm 3 benzaldehyde are added dropwise. The mixture is maintained under stirring and under hydrogen pressure of 200 kPa for 2 hours then 1.65 cm3 of an aqueous solution of 35% formaldehyde are added. The agitation under hydrogen pressure is pursued for 2 hours more then the solution is purged to nitrogen, filtered on clarcel before being concentrated in dry under reduced pressure (20 kPa) at a temperature close to 40 ° C.

In this way, 6.06 g of 2 - acetamido 3 - ((N-ethylbenzylamino) -4-phenyl) propanoic acid (2S) is obtained in the form of a beige solid which decomposes at 160 ° C.

Preparation of 2-amino 3 - ((N-methyl-amino) -4-phenyl) -propanoic acid dihydrochloride (2S): To a suspension of 6.60 g of 2 - acetamido 3 - ((N-methyl-benzylamino) 4-phenyl) ) propane (2S) in 4.80 cm3 of water at 0 ° C is added with stirring 9.60 cm3 of an aqueous solution of hydrochloric acid at 36%. The solution thus obtained is refluxed for 2.5 hours, returned to 20 ° C, then cast on a solution of 0.16 g of palladium on carbon at 5% in 1.60 cm3 of water. the obtained mixture is purged three times to the nitrogen then three times to the hydrogen before being put under a pressure of hydrogen of 100 kPa under vigorous agitation at a temperature close to 50 ° C for 2 hours then returned to 20 ° C, filtered on clarcel, rinsed 2 times in 5.00 cm3 of water, before being concentrated in dry under reduced pressure (20 kPa) at a temperature close to 40 ° C. 12.90 cm3 of 2-propanol are added to the dry extract. The suspension thus obtained is refluxed for one hour, returned to a temperature close to 20 ° C, filtered, rinsed in 3.00 cm 3 of squeezed 2-propanol then dried under reduced pressure (20 kPa) around 40 ° C. .

This gives 1.11 g of 2-amino 3- (4- (N-methylamino) 4-phenyl) propanoic acid dihydrochloride (2S) in the form of a beige solid which decomposes at 280 ° C. (ee = 96.5% by liquid phase chromatography on a chiral stationary phase composed of a crowned ether impregnated on silica (CROWNPAK CR®)).

Example 3. Preparation of 2-amino 3- (4-nitro phenyl) propanoic acid (2S): To a stirred solution of 33.37 g of (L) -phenylalanine in 56.1 cm3 of 95% sulfuric acid and cooled At -15"C, 9.95 cm3 of nitric acid is added dropwise to 100% maintaining a temperature of the mixture close to -16 ° C. At the end of the addition, the solution is maintained at -16 ° C. ° C for 2.5 hours, 250 cm3 of water and are added dropwise leaving the solution to return to a temperature close to -10 ° C, then 180 cm3 more or less of an aqueous solution of soda to 30% are added drop by drop until pH = 4.70 leaving the solution to return to a temperature close to 25 ° C. The precipitate obtained is filtered, recovered by 350 cm3 of water, placed at a temperature close to 50 ° C for 0.5 hours, returned to a temperature close to 20 ° C, filtered, squeezed then dried under reduced pressure (20 kPa) around 40 ° C. 24.35 g of 2-amino 3- (4-nitro phenyl) propanoic acid (2S) are thus obtained in the form of a white solid which decomposes at 320 ° C.

Preparation of 2-acetamido-3- (4-nitro-phenyl) -propanoic acid (2S): To a suspension of 19.9 g of 2-amino 3- (4-nitro phenyl) propanoic acid (2S) in 78.6 cm3 of water at 20 ° C is added, with stirring, 20.9 cm3 of an aqueous solution from soda to 30%. The mixture is kept under stirring at a temperature close to 20 ° C for 30 minutes then cooled to 5 ° C. To this solution, 13.7 cm3 of acetic anhydride are added dropwise, keeping an internal temperature close to 5 ° C. At the end of the addition, the mixture is obtained at 5 ° C for 0.25 hours then 9.5 cm3 of a 30% aqueous solution of soda are added until complete solubilization. To this solution are added dropwise 26.0 cm3 of a 36% hydrochloric acid solution leaving the temperature rise to 15 ° C. At the end of the addition the suspension is stirred for 0.5 hours at 15 ° C, filtered, squeezed, then dried under reduced pressure (20 kPa) around 40 ° C. thus, 19.5 g of 2-acetamido 3- (4-nitro phenyl) propanoic acid (2S) is obtained in the form of a white solid which decomposes at 191 ° C. 2-Amino 3- ((N-methylamino) -4-phenyl) propanoic acid (2S) can be obtained from 2-acetamido 3- (4-nitro phenyl) propanoic acid (2S), as described in example 3 Example 3. Preparation of methyl 2-amino 3- (4-nitro phenyl) propanoate hydrochloride (2S): a suspension of 177.4 g of 2-amino 3- (4-nitro phenyl) propanoic acid (2S) in 1800 cm3 of methanol 0 ° C is added dropwise, with stirring, 69.8 cm3 of thionyl chloride maintaining the temperature of the mixture between 0 and ° C. At the end of the addition the mixture is refluxed for 6 hours. Then 900 cm 3 of 2-propanol are added to the reflux mixture and then 1,600 cm 3 of methanol are distilled off. The suspension thus obtained is returned to room temperature, maintained for one hour in the environment under agitation, filtered, rinsed by 180 cm3 of 2-propanol, squeezed then dried under reduced pressure (20 kPa) around 40 ° C.

This gives 188.1 g of methyl 2-amino 3- (4-nitro phenyl) propanoate hydrochloride (2S) in the form of a white solid melted at 218 ° C.

Preparation of methyl 2-ethanamido 3- (4-nitro phenyl) propanoate (2S): to a suspension of 186.1 g of methyl 2-amino 3- (4-nitro phenyl) propanoate hydrochloride (2S) in 930 cm 3 of tetrahydrofuran at 20 ° C under stirring are added 83 cm 3 of acetic anhydride. The mixture is returned to 5 ° C then 241 cm 3 of triethylamine are added dropwise in such a way that the temperature of the reaction mixture is maintained below 20 ° C. At the end of the addition the mixture is kept under stirring at 20 ° C for one hour then cooled to 5 ° C. At 5 ° C 460 cm3 of water are added. The mixture is returned at 20 ° C, stirred for 30 minutes at this temperature then decanted. The lower aqueous phase is decanted and the upper organic phase is poured over 2300 cm3 of water at 20 ° C. The mixture is refluxed and 600 cm 3 of tetrahydrofuran are distilled. The obtained suspension is returned to 20 ° C under agitation, filtered rinsed three times for 200 cm 3 of squeezed water then dried under reduced pressure (20 kPa) around 50 ° C.

This gives 171.2 g of methyl 2-ethanamido 3- (4-nitro-phenyl) propanoate (2S) in the form of a cotton-wool white solid melted at 126 ° C.

Preparation of methyl 3- (4-amino phenyl) 2-ethanolamido propanoate (2S): To a suspension of 170.0 g of methyl 2-ethanolamido 3- (4-nitro-phenyl) propanoate (2S), 417.5 g of zinc powder and 850 cm3 of methanol at 10 ° C under stirring, an solution of 341.6 g of ammonium chloride in 850 cm3 of water in such a way that the temperature of the mixture is maintained below 25 ° C. At 25 ° C, the suspension is filtered rinsed with 850 cm 3 of methanol then rinsed by 450 cm 3 of water. The filtrate and the two washings are combined, refluxed to fractionate the entire methanol then returned to room temperature. At 20 ° C they are added for 30 minutes then decanted. The lower organic phase is stripped, dried over anhydrous sodium sulfate. After filtration of the sodium sulphate the solution is concentrated at 30 ° C under reduced pressure (20 kPa) then dried under reduced pressure (20 kPa) around 25 ° C.

Thus, 123.4 g of methyl 3- (4-amino phenyl) -2-ethanolamido propanoate (2S) are obtained in the form of a yellow solid which melts at 141 ° C.

Preparation of methyl 2-ethanamido 3- (4-methanoamidophenyl) propanoate (2S): To 153 cm 3 of acetic anhydride at 0 ° C, 76.7 cm 3 of formic acid are added dropwise with stirring. The solution is then heated to 50 ° C for 2 hours then returned to 0 ° C, at which temperature 150 cm 3 of tetrahydrofurane are added. To this mixture is added a solution of 148.6 g of methyl 3- (4-amino phenyl) 2-ethanolamido propanoate (2S) in 1500 cm 3 of tetrahydrofuran in such a way that the temperature of the reaction mixture is maintained below 10. ° C. At the end of the addition, the mixture is allowed to return to 25 ° C, then 1500 cm3 of water is added. The mixture is refluxed, the tetrahydrofuran is fractionated, then the mixture is returned to 25 ° C and maintained at this temperature for 3 hours. The suspension obtained is filtered, rinsed by 150 cm3 of water, squeezed, dried under reduced pressure (20 kPa) at 40 ° C.

Thus, 123.5 g of 2-ethanolamido 3- (4-methanolamido phenyl) propanoate demethyl (2S) are obtained in the form of a beige solid which melts at 126 ° C.

Preparation of methyl 2-ethanamido-3 ((4-N-methyloamines phenyl) propanoate (2S): To a solution of 100.0 g of methyl 2-ethanamido 3- (4-methanamido phenyl) propanoate (2S) in 3000 cm 3 of tetrahydrofuran at 20 ° C under stirring is added under nitrogen 378 cm 3 of a 2M solution of borane methyl sulfur in the tetrahyd ofuano in such a way that the temperature of the reaction mixture is maintained below 25 ° C. At the end of the addition, the stirring of the reaction mixture is maintained 1 hour at 25 ° C then 1000 cm 3 of methanol are added dropwise. At the end of the methanol addition, the mixture is stirred at 25 ° C under nitrogen for 16 hours then concentrated under reduced pressure (20 kPa) at a temperature of 40 ° C. At the end of the concentration, it is allowed to return to 25 ° C under nitrogen then 1000 cm 3 of dichloromethane and 1000 cm 3 of an aqueous solution saturated in sodium chloride are added, the mixture is stirred 30 minutes at 25 ° C then decanted. The lower organic phase is stripped and dried over anhydrous sodium sulfate. After filtering the sodium sulphate, the solution is concentrated at 30 ° C under reduced pressure (20 kPa).

Thus, 95.1 g of 2-ethanamido 3- (4-N-methyloamino phenyl) propanoate demethyl (2S) are obtained in the form of a white solid melting at 151 ° C.

Preparation of 2-amino 3 - ((4-N-methyl-amino-4-phenyl) -propanoic acid dihydrochloride (2S): a tan, g of 2-ethanamido 3- ((4-N-methyl-amino-4-phenyl) propanoate of methyl (2S) in 285 cm3 of water and 750 cm3 of hydrochloric acid to 36% is refluxed for 5 hours then concentrated.To the concentrated mixture cooled to 80 ° C, 900 cm3 of 2-propanol is added. It is kept at 80 ° C for 2 hours then returned to room temperature.The precipitate is filtered, rinsed twice by 200 cm3 of 2-propanol, then squeezed under reduced pressure (20 kPa) around 40 ° C.

Thus, 77.5 g of 2-amino 3- ((4-N-methyl-amino-4-phenyl) propanoic acid dihydrochloride (2S) is obtained in the form of a white solid which melts at 210 ° C.

USE OF 2-AMINO ACID 3- (4- (N-METHYLAMINO) ENILO) PRQPA QICQ. Preparation of pristinamycin IB (4-methyloamino-des (4-dimethylamino) pristinamycin IA). A culture of the SP92:: pVRC508 strain in a production medium with 16-hour additions of 1 ml of a 10 g / 1 solution in the acid water is carried out at the scale of 60 erlenmeyers, as described below. 2-amino 3- (4- (N -methylamino ') -phenyl) -propanoic acid (2S). At the end of 40 h of culture, the 1.8 liters of the resulting sample of 50 Erlenmeyer flasks are extracted by 2 volumes of a mixture of 66% 100 nM phosphate buffer of pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted 2 times with 0.5 volume of dichloromethane. The chloromethylene phases are washed with water then combined, dried over sodium sulphate and evaporated. The dried extract is taken up again by 20 ml of dichloromethane and injected onto a silica column (30 g) raised in dichloromethane and eluted successively by levels of 0 to 10% methanol in dichloromethane. The fractions containing pristinamycin IB are regrouped and concentrated in dry. The dry residue is retaken by 6 ml of a mixture 65% water and 35% acetonitrile and injected onto a column of semi-prepared Nucleosil 7 μ C8 10 x 250 mm (Macherey Nagel) eluted in a mixture of 65% buffer of 100 mM phosphate pH 2.9 and 35% acetonitrile. The fractions containing pristinamycin IB are regrouped and extracted by a volume of dichloromethane. The organic phase is washed with water, dried over sodium sulfate then concentrated dry. 52 mg of pristinamycin IB is obtained.

R.M spectrum: N: 1 H (400 Mhz, CDC13, d in ppm, ref TMS): p, 71 (dd, J = 16 and 6 Hz, 1H, 5 ß2); 0.92 (t, J - 7.5 Hz, 3H: CH3 2?); from 1.10 to 1.40 (mt, 2H: 3 ß2 and 3? 2); 1.34 (d, J = 7.5 Hz, 3H: CH3 1?); from 1.50 to 1.85; (mt 3H; 3 vi and CH2 2 ß); 2.03 (mt, 1H, 3 ßl); 2.22 (mt, 1H, 5 02); 2.33 (broad d, J = 16 Hz, 1H: t dl); 2.40 (d, J = 16 Hz, 1H, 5 ßl); 2.82 (mt, 1H: 5 e2); 2.81 (s, 3H: 4 NCH3 in for phenyl); 2.90 (dd, J-12 and 4 Hz, 1H: 4 ß2); 3.29 (s, 3H: 4 NCH3); from 3.20 to 3.45 and 3.60 (2 ts, 1H each: CH2 3 d)); 3.40 (t, J = 12 Hz, 1H: 4 ßl); 4.57 (dd, J = 7 and 8 Hz, 1H, 3 a); 4.75 (broad d J = 13 and 7 Hz, 1H: 81); 4.83 (mt, 1H: 2a), 4.89 (broad d, J = 10 Hz, 1H: la), -5.24 (dd, J-12 and 4 Hz, 1H: 4 to 5.32 ( wide d, J * 6 Hz, 1H: 5 a); 5.89 (d, J-9 Hz, 1H: 6 a); 5.90 (q wide, J = 7.5 Hz, 1H: lß); 6.53 (d, J 9 Hz, 1H: NH 2); 6.53 (d, J = 8 HZ, 2H: 4e); 7.03 (d, J = 8 Hz, 2H: 4d); from 7.10 to 7.35 (mt, 5H: aromatic H 6); 7.46 (mt, 2H: 1 H5 and 1"H4), 7.85 (dd, J = 5.5 and 2 Hz, 1H, 1 H6), 8.44 (d, J = Hz, 1H: NH 1); 8.76 (d, J = 9 Hz, 1H: NH 6); 11.63 (s, 1H: OH).

Culture of strain SP92:: pVRC508.

The mutant SP92:: pVRC508 has been cultivated in liquid production medium. The fermentation has been carried out as follows. 0.5 ml of a spore suspension of the precipitated strain is added under sterile conditions to 40 ml of the inoculum medium in a 300 ml erlenmeyer chicane. Incolum medium consists of 10 g / 1 of Corn Steep, 15 g 1 and sucrose, 10 g 1 (NH4) 2S = 4, 3 g / l NaCl, 0.2 g / 1 and 1 MgS04-7H20 , 25 g / l of CaCO3. The pH is adjusted to 6, 9 by the soda before the introduction of calcium carbonate. Erlenmeyers are stirred for 44 hours at 27 ° C on a rotary shaker at the speed of 325 rpm. 2.5 ml of the previous aged culture of 44 hours are added sterilely to 30 ml of production medium in a 300 ml Erlenmeyer flask. The production medium consists of 25 g / 1 of soy flour, 7.5 g / l of starch, 22.5 g / l of glucose 3.5 g / l of fodder yeast, 0.5 g / l of zinc sulfate and 6 g / 1 calcium carbonate. The pH is adjusted to 6.0 by the hydrochloric acid before the introduction of calcium carbonate. The erlenmeyers are stirred at 27 ° C on a rotary shaker at the speed of 325 rpm.

Claims (3)

.

1. A method of preparing an enantiomer form of 2-amino-3- (4-alkylamino-phenyl) propanoic acid of general formula. To the NH, wherein Alk represents an alkyl radical containing 1 to 2 carbon atoms in the (S) or (R) form, as well as its salts, characterized in that from respectively (L) -phenylalanine or (D) ) -phenylalanine nitration is performed, then transformation of the nitro radical of the 4-nitro phenylalanine obtained, of general formula: NH "R in which R is a hydrogen atom or a protective radical, in an alkylamino radical, after the protection in the case of the amino function of phenylalanine.

2. Use of the enantiomer form of 2-amino acid 3- (4-Alkylamine-4-phenyl) propanoic, for the preparation of biologically active product.

3. Use of the enantiomer form of 2-amino 3- (4-alkylamino phenyl) propanoic acid, according to claim 2, for the preparation of pristinamycin IB or the corresponding ethylamino derivative.